Over the course of time, the impact of HIV infection has reached worldwide proportions. Throughout the world it is estimated that there are approximately 40 million infected people, with a constantly growing incidence of new infections certified in 5 million new cases per year (UNAIDS-AIDS Epidemic Update-2005). The impossibility of carrying out control mechanisms for the infection, or their ineffectiveness, has already determined horrifying pictures in certain areas of the globe. For instance, in certain regions of sub-Saharan Africa (the region of the world most affected by the pandemic) it is estimated that more than 50% of working-age subjects are seropositive, with obvious repercussions on the economies of this area of the world already devastated in any case. But a similar course is also occurring in areas that are experiencing periods of impressive economic growth, such as the Indian subcontinent and South-East Asia. In these areas the number of seropositives is close to 10 million, with an annual exponential increase of new cases (UNAIDS-AIDS Epidemic Update-2005).
The antiretroviral therapy, where available, has improved the infected subjects' life expectancy. However it is linked to a series of adverse factors that within a few years could reduce its positive impact. First of all, today's available therapies are not curative; in other words, treated subjects fail to eliminate the virus completely, but remain infected and thus exposed to the risk of developing the severe clinical forms of infection (evident AIDS), and anyway of transmitting the infection to other people. Furthermore, available drugs are burdened with very serious side effects. This causes patients' low compliance with the therapy itself. Low compliance, along with the high molecular adaptability of the virus and other factors, has favoured the onset and spread of completely drug-resistant HIV isolates. Furthermore, the high cost of the therapy makes its use absolutely unlikely on a large scale in the world's depressed areas that, as mentioned above, represent the true explosive reservoir of infection at global level.
Overall, the above described factors make the necessity of working out alternative or, at worst complementary, strategies to the therapy of overriding importance: in the scope of HIV infection control, the development of an effective vaccine approach would certainly have a priority role.
Unfortunately, HIV infection is still nowadays an open challenge and difficult to solve by the scientific community. In fact, traditional vaccine approaches, based on administration of viral particles that are unable to infect but able to stimulate the immune system, have been demonstrated as completely ineffective towards a virus that uses molecular polymorphism, i.e. the ability to mutate in order to escape the immune response, as its own winning weapon (McMichael J., (2006) Annu. Rev. Immunol. 24: 227-55).
In the following description, after having briefly illustrated the main immunological mechanisms stimulated by HIV, the various main vaccine strategies followed up to now will be synthetically reviewed, dwelling in particular on the strategy followed in the approach underlying the invention which is the subject of the present patent application, i.e. the attainment and administration of anti-idiotype mimotopes.
The Immune Response Directed Against HIV
The natural history of HIV infection witnesses uncontrolled replication of the virus within early weeks after the infection, with viremia levels that often exceed values of 107 virus particles per ml of plasma within the first 21 days. This first stage is followed by another characterized by a sudden decrease in viremic levels, due to activation of specific response mechanisms, represented by cytotoxic CD8+ T cells and production of neutralising anti-bodies. This stage demonstrates how the immune system, once stimulated, is able to at least partially control the infection. However, the main concern is represented by, as mentioned in the previous section, the hypervariability of certain viral proteins, which enable the virus to overcome the stimulated defenses and thus resume replication in an uncontrolled manner. In the case of HIV infection, the immune system is thus continuously called upon to run after the virus variability: however, the natural history of the infection demonstrates that the immune system results constantly defeated in this chase.
The main object of an effective prophylactic vaccine would then be to present the virus, since the first contact, with an immune response able to block it and thus prevent its uncontrolled propagation in the organism.
Surface HIV proteins (gp120 and gp41), that make target cell infection possible, represent the main targets of the immune response and in particular of humoral-type immune response. Antibodies generated in the course of a natural infection however assure a protection that is limited to the virus that has stimulated such a response. In other words, those very antibodies will not be able to protect the patient who produced them from new viral variants that will develop, and (the most important fact from the vaccine point of view) all the more so would not prove protective for other patients infected by other HIV variants.
The study of HIV-induced antibody response has however evidenced a possible role played by antibody clones with broad neutralising activity against a large panel of virus isolates (Pantophlet R. and Burton D. R., (2006) Annu. Rev. Immunol. 24: 739-769). Still, only a few patients are able to produce antibodies with similar features, that will allow them to control the infection for a much longer period, slowing down progression towards evident AIDS (Braibant M. et al., (2006) AIDS. 20: 1923-30). These patients are defined by the scientific community as long-term non-progressors (LTNP) and certainly represent an example of anti-HIV immune response to be studied for the development of an effective vaccine.
In the light of the collected data, the scientific community has at this point almost universally recognized that an effective prophylactic or therapeutic vaccine should be able to stimulate an adequate antibody response, analogous to that described in LTNP patients. The main target of a potentially effective humoral response is represented by, as previously mentioned, the two surface glycoproteins (gp120 and gp41) that, in the form of trimers, compose the spikes that enable the virus to bind and penetrate within target cells. However, in particular, conserved key epitopes, and thus common to the different virus isolates, should be detected within these proteins. Laboratory and clinical experimental data have actually proved that the broad neutralising activity response is indeed directed against these epitopes (Braibant M. et al., (2006) AIDS. 20: 1923-30). The scientific community unanimously recognizes that the presence of antibodies with similar features at the time of the first contact with the virus most probably would be able to neutralise the infection, attaining what not even the most effective therapeutic protocol has succeeded in obtaining yet, i.e. complete virus clearance (Pantophlet R. and Burton D. R., (2006) Annu. Rev. Immunol. 24: 739-769).
Viral Escape Mechanisms
The selective pressure exerted by cellular and humoral immune response against HIV virus has been studied in great depth in literature, although its effects on the progression of immunological deficiency and hence on clinics are not clear. The selective pressure effected by neutralising antibodies is indeed easily observable both in vitro and in vivo since the early infection stages. In fact, the virus escapes through a series of mutations that make the usually generated non-broad range neutralising antibodies useless. Such mutations, that sometimes involve a single amino acid residue, typically implicate the surface glycoproteins and in particular the so-called gp120 protein V (variable) regions. Therefore, it is easily understandable that this key antigen's sequence variability is one of the main reasons for the inability of the antibodies generated in the course of natural infection to completely block the virus. In this context, the molecular reasons of the failure of all “classical” attempts at obtaining a protective response by immunizing subjects with whole virus particles or recombinant gp120 are likewise easily understandable (Pantophlet R. and Burton D. R., (2006) Annu. Rev. Immunol. 24: 739-769).
However, HIV defence mechanisms are not limited to the strategy, already effective in itself, of hypervariability. Structural studies of the virus have in fact revealed how HIV exploits its own hypervariable regions in order to hide key epitopes from the immune system too, such as the CD4-binding site and the so-called gp120 coreceptor-binding site, i.e. the protein portions that physically bind the target cell receptor and coreceptors at the time of infection. In other words, the virus would only expose these key regions at the time of direct interaction with the target cell, thus limiting their exposure to the immune system.
Also, HIV has developed another strategy of hiding gp120 most important epitopes: 50% of the molecule is actually covered with carbohydrates, that make the protein surface practically “invisible” to the immune system. In vivo the virus can also modify the positions of this glucidic coating, thus leading to the hypothesis of a dynamic evolution model, the so-called glycan shield. HIV would actually be able to modulate glycosilation, continuously adapting to the kind of immune response that from time to time it has to contrast. Therefore, the ability of escaping the immune response is not a widespread phenomenon, an inherent feature common to all virus particles, but a specific and continuous adaptation to the neutralising antibody response that is stimulated each time.
gp120 as a Potential Vaccine Target
As disclosed in the two previous sections, gp120 represents the main target of HIV virus-neutralising immune response. However, in the previous sections, the molecular reasons why classical vaccine approaches, even though contemplating the use of an antigen so important to the virus, did not lead to positive results have been pointed out. In particular, the use of inactivated whole virus particles, or of gp120 recombinant monomeric forms, leads to stimulation of an immune response merely limited to the virus used in the vaccine protocol, or from which the recombinant protein had been obtained. For instance, the stimulated response turned out to be limited to HIV isolates adapted in laboratory to grow on immortalized T cell line cultures. Instead, no antiviral effect was seen against “primary” virus isolates, i.e. isolates directly derived from infected patients.
The failure of these approaches has led to investigation of possible alternative routes that will be briefly disclosed in this section of the specification, and can be synthetically divided into two groups.
a) Development of gp120 Trimeric Preparations which Represent the Protein Structure Displayed on HIV Spikes Better.
This approach relies on administration of gp120, no longer in monomeric form but in heterotrimeric form, in association with the other viral surface glycoprotein, the gp41 protein. The principle at the root of this strategy is based on the observation of the different anti-genic features of monomeric gp120 compared to the trimeric forms (Pantophlet R. and Burton D. R., (2006) Annu. Rev. Immunol. 24: 739-769). However, the first data collected from approaches regarding this strategy have revealed a series of problems tightly connected with each other, both from the technical point of view and from the point of view regarding the effectiveness of the approach itself. From the technical viewpoint, the main obstacle to be overcome consists indeed in the ability of obtaining stable heterotrimeric forms that are best able to mime the organisation of the viral envelope spikes. On HIV surface, in fact, gp120-gp41 interactions are mediated by non-covalent interactions essential in order to give the overall structure of the spike the indispensable structural adaptability that characterizes its function. In laboratory, it has proved particularly difficult to obtain such molecules, which on one hand should be stable enough not to dissociate into single monomers, and on the other hand should however be able to display critical portions of the proteins, and particularly of gp120. It has therefore been necessary to adopt a series of technical stratagems in order to stabilize the trimers (mutation into the original polyprotein cleavage sites, insertion of cysteine residues into unimportant portions of the structure), or to display them onto structures as similar as possible to the viral envelope (inclusion into proteoliposomes, expression on virus-like particles). However, none of the approaches followed have allowed for completely solving the trimer stabilisation and purification problems, nor have they led to definitely superior results compared to those obtained with monomeric forms of gp120, in terms of efficacy and especially of the extent of the neutralising activity. The results obtained with approaches contemplating the use of gp120 recombinant forms mutated in order to make key portions of the protein more available to the immune system were similarly unsatisfactory.
b) Development of Innovative Epitope-Based Vaccines i.e. Based on Exposure to the Immune System of Conserved, Therefore Potentially Protective, Portions of gp120.
This group is connected with the anti-idiotype mimotope strategy, on which the invention illustrated in the present patent application is also based.
The main problem with all the above illustrated strategies concerns the inability to stimulate, in addition to a type-specific neutralising response, a broad range neutralising response. The so-called epitope-based approaches must be considered within the sphere of the attempts to reduce the type-specific response and enhance the cross-neutralising one.
To better understand the strategies connected with this group, it is useful to make a brief reference to the gp120 antigen structure. Based on comparative sequence analysis, the study of gp120 reveals, as regards the glycoprotein, 5 conserved (C1-C5) and 5 variable (V1-V5) segments. Further studies have demonstrated that the C1 and C5 regions are probably engaged in the contact with gp41, as it has been evidenced that antibodies directed against this region only recognize monomeric forms of gp120, not the trimeric ones. Instead, certain portions of the C2, C3 and C4 regions possibly form a hidden and relatively hydrophobic nucleus within the gp120 molecule, probably involved in CD4 receptor recognition. Unlike conserved regions, the variable regions (particularly V1, V2 and V3) are well exposed and accessible on the protein.
The epitope-based approaches in fact attempt to exploit gp120 structural features in order to obtain molecules that are able to target the immune response exclusively, or predominantly, to its key epitopes. In this context, one strategy has been to use gp120 monomers, from which the V1, V2 and V3 regions had been removed, in order to expose the conserved CD4-binding portions to the immune system. A similar approach has not yet given satisfactory results, also because the removal of such large portions from the protein has inevitable effects on its whole conformation and hence on the CD4-binding portion that might lose its own distinctive features.
Another possible strategy is to exploit, to the immune system's advantage, one of the HIV escape mechanisms previously disclosed, i.e. hyperglycosylation of gp120 portions. In this connection, artificially glycosylated gp120s have been obtained in laboratory, in order to hide non-protective sites and target the response exclusively to the protein's important portions. However, the results obtained with this approach have been unsatisfactory, in as much as this strategy, although leading to a reduction in the immune response directed against non-conserved portions of the molecule, has not been able to cause an extensive response to gp120 crucial portions.
At this point, it is useful to recall that, in some rare cases and in very low titres, in the course of certain natural infections, antibodies capable of neutralising a broad range of viral isolates are generated. Such antibodies (extremely rare and precious from the scientific point of view) thus represent an ideal template for an extremely targeted epitope-based approach. Exploiting the idiotype of these molecules (i.e. the antibody portion that specifically recognizes and binds the antigen), it is possible, by using a reverse vaccinology approach, to obtain other (anti-idiotype) antibody molecules which are specifically directed against the idiotype of broad range neutralising antibodies, and thus able to mime the key epitopes that they recognize. In other words, well designed anti-idiotype antibodies may represent an artificial antigen unrivalled in laboratory, as they are able to expose just the neutralising antibody-recognized key epitope to the immune system.
The analysis of prior scientific and patent literature has allowed for pointing out that anti-idiotype-based strategies have already been applied to HIV infection.
However, as far as the inventors know, the majority of prior literature relates to anti-idiotype antibodies obtained using, as the cloning template, non-human derived, especially mouse-derived, antibody molecules, i.e. antibodies obtained immunizing laboratory mice with recombinant gp120. As it has been many times scientifically demonstrated that one identical epitope may be able to stimulate a specific antibody response in an experimental animal but not in human beings, the choice of using non-human derived polyclonal preparations or monoclonal antibodies as the template for obtaining anti-idiotype antibodies makes the attainment of anti-idiotype antibodies useful for vaccine purposes in man uncertain; i.e. it is uncertain that they would be capable of effectively miming fundamental gp120 antigen portions recognized by the human immune system and of accordingly being able to stimulate an effective immune response in human beings.
Human-Derived Template Antibodies
However, a few prior documents describe human monoclonal antibodies with neutralising activity, which in some cases are proposed as the template for obtaining of anti-idiotype molecules. The majority of such prior documents, however, do not describe in practical terms the production of anti-idiotype molecules, nor their properties and applications.
The inventors are acquainted with only one prior patent document in which the achievement of anti-idiotype antibodies starting from human-derived template antibodies is concretely disclosed. It is International Patent Application WO 92/15885, published on Sep. 17, 1992. This patent application discloses a method for selection of anti-idiotype monoclonal antibodies useful for vaccine purposes for the prophylactic or therapeutic treatment of HIV infections. In brief, this method contemplates the attainment of anti-idiotype monoclonal antibodies (G1-Ab2s) using as the template a polyclonal preparation of whole anti-gp120 human Igs (Ab1s), the subsequent selection of a subset of anti-idiotype monoclonal antibodies (G2-Ab2s) characterized by the ability to react with in vitro multiple HIV strain-neutralising anti-gp120 antibodies, and the selection of a further subset of anti-idiotype monoclonal antibodies (G3-Ab2s) capable of generating, in a primate host, an anti-anti-idiotype antibody (Ab3) response, which antibodies react with the gp120 antigen and have HIV neutralising properties.
The procedure described in the WO 92/15885 application shows several disadvantages. First of all, by using whole immunoglobulins as the template, a panel of anti-immunoglobulin monoclonal antibodies are obtained which are mostly directed towards useless portions of the template immunoglobulins, i.e. the portions outside the idiotype, and which for the most part are therefore not true anti-idiotypes. Furthermore, the neutralising response achieved in primates disclosed in this patent application is weak and requires prior purification of the antibodies used in the immunization. It can thus be concluded that the antibodies obtained with the WO 92/15885 method, besides not being sufficiently specific to the useful portion of the anti-gp120 immunoglobulins (the idiotype), are not able to evoke a strong neutralising immune response (low antibody titres) and thus are not particularly promising as vaccines.